Subtractive hybridization is a process that is commonly used in association with cloning of cDNA derived from mRNA extracted from particular cells that are under investigation and is most useful for producing DNA hybridization probes that can be utilised as screening agents to detect or locate DNA, in clone colonies or cDNA libraries for example, related to genes that are differentially expressed as compared with genes of other cells that exhibit different gene expression characteristics. This technique may, for example, be used in cancer research for comparing the gene products of tumour tissue cells with those of corresponding normal tissue cells in order to study the genetic changes that have occurred at the nucleic acid level. Probes obtained using this technique which are specific to DNA whose expression characteristics are modified by such genetic changes may be useful not only for carrying out genetic screening in connection with cDNA cloning, but also as diagnostic tools.
In a typical procedure for applying this technique of subtractive hybridization to investigate differences in the active genes of a certain sample of test or target cells, e.g. from tumour tissues, as compared with the active genes of a sample of reference cells, e.g. cells from corresponding normal tissue, total cell mRNA is extracted (using conventional methods) from both samples of cells. The mRNA in the extract from the test or target cells is then used in a conventional manner to synthesise corresponding single stranded cDNA using an appropriate primer and a reverse transcriptase in the presence of the necessary deoxynucleoside triphosphates, the template mRNA finally being degraded by alkaline hydrolysis to leave only the single stranded cDNA. In one particular version of the technique, important in the context of the present invention, care is taken to avoid unwanted synthesis of any second strand cDNA in this initial stage. The single stranded cDNA thus derived from the mRNA expressed by the test or target cells is then mixed under hybridizing conditions with an excess quantity of the mRNA extract from the reference (normal) cells. The latter is herein generally termed the subtractive hybridization "driver" since it is this mRNA or other single stranded nucleic acid present in excess which "drives" the subtraction process. As a result, cDNA strands having common complementary sequences anneal with the mRNA strands to form mRNA/cDNA duplexes and are thus subtracted from the single stranded species present. The only single stranded DNA remaining is then the unique cDNA that is derived specifically from the mRNA produced by glenes which are expressed solely by the test or target cells.
To complete the subtraction process and to use the single stranded unique cDNA, e.g. for producing labelled probes that may perhaps be used for detecting or identifying corresponding cloned copies in a cDNA clone colony (labelling of such probes is frequently introduced by using labelled deoxynucleoside triphosphates in synthesis of the cDNA), in the basic subtractive hybridization technique the common mRNA/cDNA duplexes are then physically separated out using, for example, hydroxyapatite (HAP) or, more preferably, (strept)avidin-biotin in a chromatographic separation method. After this operation, one or more repeat rounds of the subtractive hybridization may be carried out to improve the extent of recovery of the desired product.
From time to time various improvements in the basic technique outlined above have been introduced, including a method of chemical cross-linking subtractive hybridization (see Hampson et al (1992) Nucleic Acids Res. 20, 2899) which can enable the need for physical separation of the common mRNA/cDNA duplexes from single stranded unique cDNA to be avoided, but the process of producing labelled probes and/or cDNA subtraction libraries has still required a relatively large number of cells, especially since neither the target cDNA nor the driver RNA have been renewable. Although the introduction of PCR technology provided a new tool with a potential for enabling subtractive cloning to be applied to smaller numbers of cells and many strategies based on the hybridization of original target cDNA to so-called driver RNA or antisense cDNA have been developed to overcome the problem of limited starting cell number, problems have still remained. In particular, the methods developed have still been complex and have usually required multiple rounds of PCR and of subtractive hybridization which are liable to produce artifactual results by virtue of representational skewing during the amplification process.
One difficulty encountered in using PCR for amplifying cDNA is the fact that it cannot usually amplify efficiently full length cDNA strands corresponding to mRNA molecules from which they are derived, and when there is a mixture of cDNA strands or strand fragments covering a wide range of different lengths there is a tendency for preferential amplification to occur of the shorter length cDNA molecules. Ideally, for satisfactory PCR amplification of cDNA molecules, in order to reduce any non-uniformity and bias due to inefficient amplification of larger cDNA molecules the molecules in the reaction mixture upon which PCR amplification is performed should each have a size in the range of 100-500 base pairs, and within this range the sizes should be distributed randomly.
Obviously, it can also be important in using PCR amplification to provide amplified single stranded molecules that strand sense should be maintained.